High grade astrocytomas are the most common primary central nervous system (CNS) malignancy afflicting nearly 10,000 pediatric and adult patients each year in North America alone. While surgery remains the most effective curative therapy for CNS tumors, grade III (anaplastic astrocytoma) and grade IV (glioblastoma, GBM) astrocytomas exhibit a highly invasive histology that precludes effective surgical resection. Consequently, GBM tumors are among the most malignant cancers with a mean survival of approximately one year despite multimodal therapy with surgery, radiation and chemotherapy. In contrast to many cancers, the survival outcome of patients diagnosed with GBM has improved only marginally over the past several decades. While our understanding of the pathophysiology of these high grade malignancies has improved over the past several years, discovery of effective therapies have been limited as few novel targets affecting the complex pathways dysregulated in GBM have been identified.
Malignant astrocytomas display an impressive spectrum of genetic and epigenetic abnormalities affecting multiple pathways relevant to cell growth, survival and remodeling of the tumor microenvironment. Over the past several years, numerous genome-wide studies have demonstrated that GBM possesses a remarkable degree of heterogeneity with regard to genetic mutation, gene expression profiles, and epigenetic modifications. This degree of biologic heterogeneity has undoubtedly contributed to the challenge of identifying key proteins that are critical to the underlying pathogenesis of this aggressive disease.
Posttranslational modification of proteins is a common activity involved at virtually all levels of cellular regulation. The enzymes that covalently modify amino acids add an additional layer of regulatory control over multiple cellular processes including chromatin remodeling, gene transcription, signal transduction, DNA repair and RNA processing. The enzymes of the protein arginine methyltransferase (PRMT) family represents a group of proteins that are evolutionarily conserved amongst a wide variety of organisms. PRMT enzymes covalently modify both histone and a growing number of proteins that are critical to the maintenance of numerous cellular regulatory networks. The PRMT5 enzyme is a type II arginine methyltransferase that utilizes the donor molecule S-adenosyl-L-methionine to catalyze the transfer of a methyl group to two of three guanidino nitrogen atoms within the arginine molecule. PRMT5 drives the formation of both ω-monomethylarginine and symmetric dimethylarginine residues to affect a wide range of key biologic functions at the level of chromatin to control transcriptional repression and as an enzyme that modulates non-histone protein function.
In addition to classic gene mutations, epigenetic silencing of tumor suppressor genes (TSG) frequently leads to dysregulation of signaling pathways and promotion of tumorigenesis. Chromatin remodeling enzymes like histone deacetylase (HDAC), DNA methyltransferase, and protein arginine methyltransferase 5 (PRMT5) are involved in silencing TSG expression and over expression of these enzymes can promote cellular transformation. Drugs that inhibit HDAC enzymes (HDAC-I) or prevent DNA methylation are currently being examined in clinical trials treating patients with cancer. HDAC-I and hypomethylating agents had been shown to possess anti-tumor activity in malignant gliomas both in vitro and in vivo, however, clinical trials investigating these agents have been disappointing, pointing to the need to identify other promising epigenetic targets. PRMT5 has been shown to methylate histone proteins at H3 arginine 8 (H3R8) and H4R3, and trigger TSG silencing. PRMT5 has either a positive or negative effect on its substrates by arginine methylation when interacting with a number of complexes and is involved in a variety of cellular processes, including RNA processing, signal transduction, transcriptional regulation, and germ cell development. Recent studies showed PRMT5 to be a major pro-survival factor regulating eIF4E expression and p53 translation. PRMT5 depletion triggers p53-dependent apoptosis and sensitized various cancer cells to Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) without affecting TRAIL resistance in non-transformed cells.
Several groups have recently described how PRMT5 dysregulation can affect cell growth. Work previously reported demonstrates that overexpression of PRMT5 is involved in the pathogenesis of mantle cell lymphoma, an aggressive hematologic malignancy. While that work contributed information regarding the mechanism of PRMT5 overexpression, the functional consequences of PRMT5 inhibition on growth and survival of the transformed cell remain poorly characterized. We have found that the PRMT5 enzyme is overexpressed in cell lines derived from high grade astrocytomas and in primary anaplastic astrocytoma and GBM tumors. The degree of PRMT5 overexpression was related to proliferative index of both cell lines and primary tumors and was found to be an independent prognostic factor that could identify patients with more aggressive disease and poor overall survival. Because this enzyme is intimately involved with numerous processes that are frequently dysregulated in cancer, we were led to evaluate the consequences of PRMT5 inhibition in high grade astrocytomas as these cancers display overexpression of PRMT5 in the context of profound molecular heterogeneity. We found that inhibition of PRMT5 overexpression in GBM cells led to restoration of critical regulatory pathways affecting cell growth, survival, and tumor suppressor and immune modulatory gene expression. These findings suggested that experimental therapeutic strategies aimed at inhibiting the effects of PRMT5 overexpression in cancer might lead to a better understanding how to directly and indirectly affect GBM tumor progression.
Identification of novel therapeutic strategies to improve the outcome of patients with high grade astrocytomas has, for the most part, proved elusive to date. This is due to several problems inherent to high grade gliomas including: (1) the degree of molecular heterogeneity; (2) lack of a universal target selectively expressed in the tumor; (3) invasive nature of the disease; (4) rapid evolution of chemo and radiation resistance; and (5) likelihood that high grade gliomas arise as a consequence of cancer stem cell and clonal evolution. Identification of a central factor driving these multiple pathways contributing to growth, survival, invasiveness and resistance has proven difficult.
PRMT5 overexpression in high grade gliomas can serve as an attractive therapeutic target for several reasons. First, PRMT5 is selectively overexpressed in high grade gliomas and not in normal human astrocytes or neighboring primary brain tissue. Second, PRMT5 knock down results in cell cycle arrest, apoptosis, reduced invasiveness and restoration of tumor suppressor and immune modulatory cytokine gene expression. Third, PRMT5 is over expressed in glioma brain tumor stem cells. PRMT5 overexpression is highly relevant to high grade glioma biology and therefore fulfills the criteria of an optimal target for this and other diseases.